DNA damage occurs frequently in cells and is generated by both exogenous and endogenous agents. Of all DNA lesions, double-strand breaks (DSBs) are most dangerous to the cell, and persistent breaks can lead to genomic instability resulting in cell death or cancer. Although the mechanisms of DSB repair and the cellular response to DNA damage (DDR) have been extensively investigated, little is known about the specific cellular response to DSBs within an actively transcribed locus. DSBs in transcribed regions are especially precarious to the cell. Somatic mutations arising from aberrant repair of such breaks may inhibit the production of functional gene products or produce deleterious gene products that compromise normal cellular activities. Furthermore, persistent breaks at transcribed loci potentiate genomic rearrangements placing translocated loci under abnormal regulatory constraints or creating fusion genes, which are known to drive carcinogenesis. Therefore, it is critical that DSBs in regions of active transcription are correctl and efficiently repaired to prevent the incurrence of oncogenic mutations. We hypothesize that cells implement a modified damage response to address breaks at actively transcribed loci. Such a response likely involves coordination between DDR/DSB repair factors and the transcriptional machinery to temporarily silence genes while repair is occurring, thus preventing adverse outcomes. We aim to develop an experimental system where DSBs can be generated at transcriptionally inducible endogenous loci in repair-deficient cell lines. This novel approach will enable us to determine how local transcription is affected by the presence of a break. We predict that transcription will be temporarily repressed in response to a DSB, and that this silencing is achieved through DDR-driven targeting of the transcriptional machinery itself and/or alterations in local chromatin structure to exclude factors involved in transcription. Thus we will conduct experiments to identify mechanisms involved in regulating transcription at or near a break site. Taken together, the proposed studies will provide valuable basic science insight regarding the molecular pathways used by eukaryotic cells to maintain genomic integrity. In addition, this research will reveal signaling networks that may be altered in cancer cells and provide potential therapeutic targets for cancer treatment.